Project Summary/Abstract Because electron transfer reactions are fundamental to life processes, such as respiration, vision, and energy catabolism, it is critically important to understand the relationship between functional states of individual redox enzymes and the macroscopically observed phenotype, which results from averaging over all copies of the same enzyme, encompassing varying levels of catalytic activity. To address this problem, we will develop a bifunctional nanoelectrochemical-nanophotonic architecture - the electrochemical zero mode waveguide (E- ZMW) - that can couple biological electron transfer reactions and luminescence. The E-ZMW combines the capacity to trap electromagnetic radiation with the ability to control electrochemical potential in its sub-attoliter total volume. Single copies of redox enzyme molecules will be immobilized in E-ZMW nanopores at the surface of a metal annulus that can function both as a working electrode, controlling the potential at the enzyme, and as the optical cladding layer of a ZMW. Our first aim is to develop E-ZMW architectures capable of supporting potential controlled single molecule redox reactions with oxidoreductase enzymes. First, we will develop parallel arrays of electrochemically-active single molecule ?beakers? with functional oxidoreductase enzymes immobilized on bifunctional working electrode/optical cladding (WE/OC) layers. Then we will use the WE/OC for potential-control of enzyme redox state, and measure the effectiveness of doing so by potential- dependent fluorescence dynamics. With these capabilities in-hand, it will be possible to measure single reaction turnover events. Furthermore, the confined environment of the E-ZMW makes it possible to achieve in situ control over reaction conditions and delivery of reactants. This aim will be accomplished by elaborating the basic E-ZMW architecture to obtain a dual-electrode nanopore structure with the capacity to synthesize and deliver substrate molecules in situ and on-demand and to exploit this capability to characterize single (reactive oxygen species)-enzyme reactions and their potential dependence in situ. We anticipate that the unique capabilities developed in this project will open new avenues for coupled electrochemical and spectroscopic investigations of single enzyme molecules occurring under tightly controlled conditions. These structures will establish new experimental capabilities for fundamental enzyme biochemistry, but the basic architecture and approach should lend itself to technological applications, such as biochemical processing, as well.